DK2406679T3 - AUTO FOCUS PROCEDURE AND AUTO FOCUS DEVICE - Google Patents

Description

Description

The application relates to an autofocus method in which light from a light source is focused into a target light focus in a sample and then thrown back, and the reflected light is passed through an optical system and at least one aperture in two light paths to at least two detector elements.

State of the art

Two methods for automatically focusing microscopes on a specimen are known: • The position of a specimen or the distance from the specimen to a reference point is measured, light reflected from the specimen being examined for diagrams, intensity or the like, or interferometrically examined. • The images from the sample are examined for contrast, resolution, autocorrelation or phase contrast.

Within the microscopy, a sample usually consists of a sample material to be examined which is applied to a translucent sample holder and covered with a thin translucent cover glass. A position measurement of the sample material often leads to measurement of the position of the reflection planes at the layer boundaries of the sample. Since a reflex at the air-cover glass layer is far more powerful than a reflex at a boundary layer on the sample material, the air-cover glass reflex typically radiates the reflex suitable for an autofocus at a boundary layer on the sample material.

From US 6,130,745 it is known that the position of a strong reflex layer is measured above or below the sample and that, based on the thickness of the sample, the position of the sample material located at a known distance from the reflex layer is deduced. However, in the described test, the tolerances in the layer thicknesses (for example, of the cover glass or of the slide holder) when using high-resolution systems are typically greater than the depth of field of the imaging system and focusing cannot always be guaranteed by such a method. WO2005015120 relates to a method and apparatus for focusing detection in an optical measuring device such as a microscope. The invention is preferably suitable for detection for measuring biological or chemical samples.

An object of the invention is to present an autofocus method by which an optical system, e.g. a microscope can be quickly and accurately set focusing to a reflective layer in a sample.

Achieving the purpose

This object is achieved by a method according to claim 1.

Surfaces, e.g. interfaces, hereinafter also to be understood as layers. One of the layers is advantageously an interface. The light paths are conveniently at least partially separated from each other, in particular they are separated from each other in the optical system. The separation is conveniently carried out by means of a darkened area between the light paths. The blackout can be produced by means of an aperture.

The course can be recorded by means of punctual measurements at several positions of the target light focus, suitably separated by light paths. The radiation property of the reflected light may be the radiation intensity. The set focus position is a desired focus position in which the optical system is conveniently positioned in such a manner as to the sample that an image capture of the sample results in desired images.

In addition, the optical length of the light paths can be determined by the invention. The optical length can then be measured from the sample to an aperture before a detector. Appropriately, the optical lengths of the light paths are chosen differently. In this way, through the separate interpretation of the optical length of the light paths, a deviating signal can be generated for a selected reflective / scattering sample structure and focused on this or on a focus target plane located at a known distance to it. A layer that throws light back may be a reflective and / or scattering sample structure and may be particularly a boundary layer, especially a boundary layer, respectively. interface adjacent to the sample material.

The autofocus method is a method for automated focusing of the optical system at a desired focus position or focus target plane, e.g. within the sample. When focused on the focus target plane, the optical system can reproduce an object placed thereon sharply on an image plane in which a detector and a detector, respectively, are appropriately positioned. a camera. After autofocusing, the sample can be reproduced using a camera.

The sample may include, for the study, prepared sample material, a holder on which it is placed, and a cover glass to cover it. A layer structure that is translucent to the autofocus light and at whose layer boundaries the reflection or scatter of the autofocus light used is also suitable. The sample does not necessarily have to be translucent for the autofocus light after the layer intended for focusing. The reflection / scattering at a boundary layer described herein may also be induced by a reflecting / scattering particle layer, respectively. failure site strikes in the material. The layer boundaries can be pretreated (for example, mirrored) to increase the autofocus system signals.

The focus target plane is the plane within the sample to which the optical system is to be focused, or to which the position of the desired focus must be at a predetermined distance. The focus target plane is, appropriately, a reflection plane on which incident light is reflected. It may be a boundary layer within the sample, e.g. the plane of a glass sample material interface. Likewise, the spread may be utilized by the sample itself.

The light which is directed to the light paths is suitably derived from a common light source, in that not only is initially radiant material but also a reflective layer, aperture or the like. Typically, a one- or two-dimensional light source is used. The two light paths are suitably formed symmetrically with respect to each other and particularly symmetrical with respect to the optical axis of the optical system.

The light source, which is particularly dot-shaped or line- or strip-shaped or includes several light dots, is focused in the focus light focus of the sample through the optics. In this way, it can be reproduced in the sample. The target light focus is usually point-shaped but may alternatively be one- or two-dimensional depending on the light source shape and e.g. include several points. The target light focus is advantageously located in the focus or near the focus of the optical system to be focused. The focus of the optical system can be a focus plane. An object that is in focus in the optical system is reflected sharply by the optical system in an image plane. It is also possible that the focus light is at a preset distance from the focus of the optical system. This allows the focus light focus to be set to a reflection plane, e.g. a boundary cover glass sample material, the focus of the optical system e.g. lies at a distance of 20 μηη to the boundary layer of the sample material.

A portion is thrown back from the light hitting the specimen. Reflection can be understood as a reflection and / or a dispersion in the following. The layer that throws the light back can be a reflective and / or scattering layer. When reference is made in the following to a reflection, it should also be possible to include a spread.

The two light paths are suitably arranged symmetrically about the optical axis of the optical system. They advantageously hit the sample from different directions so that their reflexes are radiated in different directions and thus can easily be analyzed separately from one another. The detection of the individual layers in a layer structure is facilitated when the angle of the light paths striking is selected in such a way that reflections from adjacent layers do not cover each other. If a scattering layer is used to determine the focus position, the division of the light paths should take place first in the detection path. Advantageously, the light in the autofocus system has a different frequency than light that can be used for the study or the the reproduction of the sample. The light property is suitably the light intensity.

The optical system may be the optical system of a microscope. It has an optical axis usually oriented perpendicular to a sample plane in which the sample extends.

The light paths between the light source and the reflection layer, respectively. between the reflection layer and the detector can be termed illumination paths, respectively. detektionsbaner. An autofocus light path thus consists of a lighting path and a detection path. The difference in optical length can now be produced in both the illumination path, the detection path as well as in both paths. The following describes a realization in the detection path. The optical length of the webs is measured by means of at least one aperture in front of the detectors. Thus, by means of a position dependent on the optical length of the light paths at the aperture, the optical length of the system can be concluded. The following is a possible realization:

The detector elements are e.g. positioned in such a manner with respect to an element of the optical system, for example, in relation to an aperture, that the course of a radiation property detected by the detector elements is mutually different.

The element of the optical system may be an aperture, e.g. immediately in front of the detector elements, a beam splitter, a mirror or other suitable element.

If light from a light path is reflected by two layers of the sample superimposed on each other, the path of light is respectively. the optical length of light from one layer, e.g. in relation to the detector or an aperture in front of the detector, longer than the light path respectively. the optical length from the second layer. Hereby the two light paths from the two layers, e.g. relative to the detector elements, be different. Conveniently, the light paths proceed such that they are blocked in various ways by an aperture in front of the detector elements, e.g. one light path is completely or partially blocked and the other partially or not at all. In this way, the light paths can be detected individually and without another light path from another layer. Conveniently, the head reflex from a glass-air boundary layer is attenuated above the sample by means of an aperture in front of the detector elements, while the target light focus is moved by the underlying layers that reflect light of different strength. In this connection, the light from these layers can pass through the aperture. In this way, layers can be detected that reflect significantly weaker than the glass-air boundary layer.

The aperture, respectively. its aperture is located in an image plane of the optical system, that is, in a plane in which an object focused by the system is reproduced. The aperture is a depiction of the source of interception.

The light reflected from the target light focus is appropriately reproduced in the aperture plane corresponding to the shape of the target light focus. The aperture is advantageously positioned in such a way that it lets light reflected in the target light focus from both light paths, especially with the same strength. In this connection, the aperture appropriately blocks light that has been reflected above and below the target light focus, completely or asymmetrically in relation to the two light paths.

The aperture is conveniently not, as usual, symmetrically positioned around the optical axis of the optical system, but asymmetrical to the optical axis, especially asymmetrical to the optical axis of the two light paths at the aperture of the aperture. In particular, it is located completely outside the optical axis. In this way, a selection of one or the other light path can be obtained for a separate analysis at different positions of the target light focus.

Particularly precise focusing can be achieved when the processes are recorded continuously. In an advantageous embodiment of the invention, a focus is set in the optical system in such a way that the signals of the detector elements are in a fixed relationship. By a light incident on the detector elements in a fixed ratio, a position of symmetry between the light paths and thus the focus target plane can easily be detected. This can happen even more simply if the signals have the same strength. The difference between the paths of the paths is chosen in such a way that the signals in connection with an overlay of the signals overlap at one edge and thus have an intersection. At this intersection, the signals have the same strength. By zeroing the difference signal, the identical strength of the signals can be easily detected. In a further embodiment of the invention, it is arranged that a target position for a focus in the optical system is detected by means of the signals of the detector elements and the focus is set with an actuator by the registered target position. The target position may be a position read out from the actuator, e.g. the position at which the signals of the detector elements are equal. It is also possible to use this setting only as a preset. Alternatively or e.g. In addition, as a fine tuning, it is conceivable that the target position is achieved by a control where the detector signals are used as control input signals and the signal for controlling the actuator is used as a control output signal.

A simple and reliable automatic focusing can be achieved when the detector elements are calibrated in such a way that the strength of their signal evoked by light emitted from a boundary layer is identical. In this connection, the focus position is suitably in the reflective layer respectively. the layer that reflects the light. Alternatively, the detector elements may be set such that their signal strength is purposefully different, e.g. to achieve a targeted focus offset.

A good orientation can be obtained during the search for the target position of the focus or the target. the focus target plane as the target light focus moves through the focus target plane toward a sample-air interface and the sample-air interface reflex is used for a rough orientation. When examining a sample, it may be necessary to examine it in different places, e.g. if it is larger than a field of view on the microscope. To this end, after an initial examination, it is moved perpendicular to the optical axis of the optical system and then re-examined. A rapid automatic focusing after such a movement can be obtained, the signals of the detector elements after a movement of the sample perpendicular to the optical axis of the optical system can be checked for plausibility with respect to a continued coarse setting for the focus target plane. If plausibility is found, a time-consuming completely new focus can be dispensed with. Plausibility can be a limit value in relation to the difference of signals which must not be exceeded. The plausibility check can also be used for coarse setting, so that when plausibility is found, only one fine tuning is performed. In a further embodiment of the invention, it is arranged that the light source has a pattern of light reproduced in the sample. The light pattern may be one-, two- or three-dimensional and is suitably reproduced in a plane perpendicular to the optical axis of the optical system in the sample. In this connection, reflected light is recorded from several pattern points in the light pattern, each separated by light paths. Hereby, a shift of the focus target plane can be detected from several target positions relative to the various pattern points, e.g. relative to the optical axis. The signals produced in this way can be used to control the autofocusing.

The invention further relates to an autofocusing device with an optical system for focusing light in a measuring light focus in a sample and for directing light reflected from it through an aperture to at least two detector elements.

It is proposed that the autofocusing device comprises an actuator and a control device for moving an element of the optical system or sample through the actuator in such a way that the target light focus is moved in layers of the sample reflecting light of different strength, the detector elements being placed on a in such a way that the course of a radiation property detected by the detector elements in this connection is different and the control device is arranged for analyzing the courses at several positions of the target light focus. In connection with the movement of the element of the optical system relative to the sample, the actuator may move the element or sample relative to a fixed reference point, e.g. a ground surface. Advantageously, the control device is designed to control one, more or all of the above steps. Advantageously, the autofocusing device comprises a measuring system adapted to record the distance from the element of the optical system to the sample or a dependent distance, particularly in a non-optical manner. Once the focus position is found optically, the distance can be measured with the additional measurement system and maintained during illumination of the sample.

For recording color images, it is known to use a color camera with a color sensitive detector. A color sensitive detector is usually limited to three colors. When a Bayer pattern is used, one pixel in each case consists of one blue, one red and two green-sensitive detector cells, from which signals all intermediate colors are composed. With the required four detector cells per pixels, the resolution of such a color detector is low.

To obtain a high image resolution combined with a high color resolution, a line spectrograph is known. An object is scanned line by line, the image in a line being arranged spectrally in a fan shape, for example by means of a prism, so that a three-dimensional image of the line is created. In this way, a three-dimensional image is stored, line by line, and the individual images are composed into a three-dimensional color image.

In addition, it is known that several color images move consecutively in front of the detector and in this way several images of one object are sequentially recorded in different frequency ranges. The recordings can be combined into a hyperspectral image.

The object of the invention is to present a method for capturing an image of an object with which high resolution color images can be recorded.

This object is achieved by a method of capturing an image of an object, in which the object is reproduced by means of an optical system on multiple detector elements in a detector, and the image is divided into several image areas that are filtered differently, with a light filter with multiple filter areas. that filter differently. According to the invention, it is proposed that the image areas of the image, which are filtered differently, are reproduced simultaneously, especially one after the other, on the detector. There is no need to change filters in front of the detector, and recordings of the object can be made quickly in succession.

The light filter may be a spectral filter or a polarization filter. It can be located immediately before the detector or directly on the detector. Conveniently, the light filter is located in an image plane of the optical system, where 1/10 of the optical system focal length of the light filter can be tolerated as a distance from the mathematical image plane and can still be considered as being in the image plane. The light filter may be an edge filter, interference filter or absorber filter. The filter regions can be filtered differently spectrally, so that the image regions are filtered differently spectrally. They may have various concrete shapes, such as stripes, a chessboard pattern and / or a micro pattern, in which the filter regions have a length and / or a width of less than 100 μηι. Appropriately, the filter areas are larger in size than two, especially larger than ten detector elements or pixels. There is also the possibility of a filter characteristic that varies continuously in a spatial sense.

The detector may be designed as a chip and is suitably sensitive in the spectral ranges of the filter regions. It can be a CCD (Charge-coupled Device) or a Complementary Metal Oxide Semiconductor (CMOS) sensor. The detector elements are advantageously arranged as a two-dimensional grid in the form of a matrix. The detector is suitably designed as a monochrome detector and is sensitive in the spectral range of the structured filter.

The dimensions of the filter regions are advantageously adapted to the dimensions of the detector elements, for example in that a width and / or length of a filter area are each an integer multiple of a size of one of the detector elements, for example m x n detector elements. The light filter may be attached directly to the detector, for example directly on a detector chip, or may be evaporated directly on the sensitive surfaces of the chip.

The filter areas suitably correspond to a structure and / or division of the sample reproduction, respectively. of the sample or of sample glass. They may be as large as regular structural sections of the sample reproduction and / or its shape. In an advantageous embodiment of the invention, the filter regions are moved via an image pattern of the image from recording to recording of the object, such that each point of the image pattern is recorded in several light properties, especially spectra. These values are appropriately assigned to the point and can be displayed and / or saved. In this connection, the image pattern may be the whole image or a section of the image. The movement is a relative movement in that the filter regions can rest, e.g. relative to a detector housing, and the image pattern is moved, or vice versa.

By multi-shooting any point in multiple light properties, such as colors, a color image of the several shots can be composed. By dividing the light filter into the filter regions, only a small order of one dimension of a single filter area is sufficient for one movement of the light filter, so that not all the light filter has to move away from the detector and a new light filter is moved towards the detector. Due to the short stretch of movement, the movement can be performed very quickly.

In order to obtain a high light output, it is advantageous when signal contributions in the individual detector elements within a filter range that can be assigned by a movement of the sample reproduction relative to the detector in a sample region are cumulated in a value. Such cumulative values of a sample range from different filter ranges can be pooled into a total light property information for the sample range.

During the movement of the filter regions towards the detector, the image pattern can remain at rest, so that the filter regions move relative to the detector. It is also possible to move the image through the detector so that the filter areas rest relative to the detector. The movement of the image through the detector can be accomplished by movement of the optical system or part of the optical system relative to the detector. A further possibility is to move the filter and detector relative to the optical system, which rests, for example, in relation to a camera body. In general, one or two of the three elements detector, light filter and optical system can each be kept at rest relative to, for example, a camera body, whereas the remaining two or the remaining one element is movable / movable relative to the other elements. Advantageously, the optical system is part of a microscope, the housing of which is fixedly fixed to a sample table on which the object in the form of a sample can be moved, e.g. on a movable tray, in particular by means of a motorized drive and a position control by means of a control device. Advantageously, the object is moved relative to the optical system and the light filter, and the object is recorded in multiple recordings in each case as image sections, the position of the filter regions in the image sections being unchanged in each case. In this connection, the optical system can rest in a housing, for example for the microscope, and the object and with this object's light pattern are passed past the optical system, as the image sections and with these filter regions migrate over the overall image.

For example, if the object is oblong, for example in the form of a series of samples, the total object may be recorded by a large number of image areas recorded successively, each pixel of the object being recorded in many colors or in each color, respectively. through each filter area. In this way, a color image of the entire object can be captured very quickly. Movement of the color filter relative to the optical system or detector may be waived. Since a device for recording images of samples is often passed via an actuator to a controlled movement of the samples along the recording device, for example the microscope, the recording device can thereby remain held in a particularly simple manner. Conveniently, the filter regions are formed as stripes extending from one image side to the opposite image side and with respect to their longitudinal direction are oriented perpendicular to the direction of movement. Also, an extension from an image section page to the opposite image section page is sufficient. Hereby, every single pixel in the object can be passed particularly easily through all the filter areas of the light filter. Advantageously, the movement is performed such that a pixel is moved from one shot to the next shot with the width of a filter area. The width in each case is appropriately several pixels. A small overlap area, e.g. corresponding to the double accuracy of the moving actuator, is appropriate in this regard.

To achieve a particularly high resolution, especially at image sections of particular interest, it is advantageous when the motion from one image recording to the next image recording is less than one image pixel. Using the movement in the sub pixel area, a sub pixel resolution can be calculated.

The determination of motion is advantageously defined by a control device which particularly automatically detects image areas of particular interest and triggers a sub-pixel motion. The movement can be performed in different modes, e.g. a sub-pixel mode, a filter range width mode, in which the motion from one shot to the next shot corresponds to the width of a filter area, or a multipixel mode in which the motion constitutes several pixels of the detector. Controlling only two of the three modes described is also possible. In an advantageous embodiment of the invention, the light filter is an edge filter whose edge frequency varies in the spatial course of the light filter perpendicular to the edge and particularly perpendicular to the motion. In this way, the color resolution of an overall image from the recordings can be controlled through the control of the extent of the motion step from one recording to the next recording. The light filter is suitably connected to an actuator and a control device which serves to control the movement of the light filter.

In addition, it is suggested that the light filter comprises two edge filters which are arranged one after the other in the beam path of the imaging, and whose edge frequency in the spatial course of the edge filters each varies perpendicular to the edge - and especially perpendicular to the motion - with a mutually opposite frequency course. Hereby - by a corresponding placement of the edge filters relative to each other - a spatial transmission window can be produced which, when moving the edge filters relative to each other, can be enlarged and reduced both spatially and spectrally. This allows for high frequency and space variability in the recorded images.

Particularly good spectral matching of the detector and light filter can be achieved when the detector comprises several detector regions which are different in color sensitivity and each are sensitive in one color region, and at least one filter region is located in front of each detector region. .

This is advantageously adapted in its color range to the color range of the detector so that the color areas of the light filter are different. The adjustment is advantageously made in that the transmission of the filter region is within the sensitivity range of the detector region concerned and is not within a color range of one of the other detector regions.

The various detector regions may be located directly next to each other in spatial terms, for example in a continuous array of detector elements, or arranged spatially apart so that the optical system comprises one or more elements for controlling the reproduction of the object to multiple detector regions. , for example, a dichroic mirror or the like. The detector regions are advantageously operated synchronously so that the reproduction of an object on the detector regions is recorded in several color channels at the same time. In a further advantageous embodiment of the invention, it is arranged that the filter regions have different transmission values and that the transmission values are each adapted to a recording characteristic of the detector, in particular to obtain a uniform image illumination in connection with a non-constant spectral sensitivity of the detector elements. In this way, a particularly good image result can be obtained. The adjustment can be realized at a different size of the filter areas. A further possibility is to adapt a different frequency transmission width of the filter regions to the detector. Thus, a frequency transmission width may be greater in a frequency range in which the detector is less sensitive and less in a frequency range in which the detector is more sensitive.

In addition, it is possible to adapt the transmission power, that is, attenuation of the filter region, to the detector, so that a higher attenuation is selected in a frequency range in which the detector is more sensitive than in other frequency ranges.

Also, a high image quality can be obtained when the filter regions have different transmission values, and an activation of the detector elements is adapted to a transmission value of the pre-filter area that shadows it. Thus, a gain can be increased, an integration time extended, or pixels added when a filter region has a high attenuation relative to another filter region. This allows uniform illumination of the image in all frequency ranges. If the transmission value of a filter range is particularly high, it is also possible to select only every other pixel. Advantageously, a different activation of the detector elements follows a movement of the filter regions through the detector. For example, if the light filter is moved through the detector, this movement can be recorded so that the detector elements covered by the area can be assigned to each filter area. The control of the detector elements can hereby be adjusted with respect to pixels for the respective assigned filter area.

The invention further relates to a device for recording an image of an object with a detector comprising several detector elements, an optical system for reproducing the object on the detector and a light filter with several filter regions that filter differently.

Especially high resolution color images can be performed when the light filter is positioned so that multiple image areas that are filtered differently through the filter areas of the image of the object are simultaneously reflected on the detector.

The device comprises a control device advantageously arranged to control one, several or all of the above-mentioned process steps.

The invention is explained in more detail on the basis of exemplary embodiments shown in the drawings.

Referring to: FIG. 1 shows a microscope with an autofocus device in a schematic view; FIG. 2 is a diagrammatically illustrated beam path, respectively. illumination paths in the autofocus device for a sample; 3-6 reflection beam paths respectively. detecting paths from the sample to two detector elements; 7 is a diagram of signals from the detector elements and a changed working distance over a period of time; FIG. 8 is a schematic diagram of the signals with a difference signal; FIG. 9 is a projection of a bright spot on a moving skewed sample; FIG. 10 is a projection of a light source pattern on a resting skewed sample; FIG. 11 is a fragmentation of a beam passage through a semi-permeable mirror; FIG. 12 is a fragmentation of a beam passage through a dichroic mirror; FIG. Fig. 13 is a schematic view of a microscope aimed at a sample with a camera with a light filter on a detector; 14 is a beam path from a sample to three detectors; FIG. 15-18 a detector having a light filter in four different positions relative to a sample,

FIG. 19 is a diagram of an edge filter having an edge moving continuously in the filter surface; FIG. 20 is a transmission diagram resulting from two edge filters arranged one after the other; and FIG. 21 is a sensitivity diagram for a detector.

FIG. 1 shows an autofocus device 2 integrated in an optical imaging system 4. In this particular embodiment, the optical imaging system is a microscope for fluorescence analysis of biological material in a sample 6. For this purpose, the optical imaging system 4 comprises an image detector 8 and 8 respectively. a camera connected to a recording device 10 for recording control and storage of recorded images, or an eyepiece for direct observation of the sample. The control device 10 is both part of the optical imaging system 4 and of the autofocus device 2 and serves to control the autofocus method described below.

The autofocus device 2 comprises a light source 12 which provides light for the autofocus method. It can also provide the light for the fluorescence analysis, although it is generally more convenient that the optical imaging system 4 for this purpose include another light source not shown here. The light source 12 is provided with a light generator 14, e.g. an LED (Light Emitting Diode), and an optic 16 for forming the radiated light which may comprise a light scatterer. An aperture 18 having an aperture pattern produces a one- or two-dimensional light source pattern which is suitably symmetrical with respect to an optical axis 20 of an optical system 22 which, in addition to the optics 16, may comprise additional optical elements 24 and an objective 26 in the optical imaging system 4. A spatially defined light source may also replace elements 16 and 18. A means 28 corresponding to an aperture divides the sample 6's light from the light source 12 into several light paths extending apart from the means 28 to sample 6 and into the sample. 6 is brought to a common target light focus (lighting paths). Alternatively, the means 28 may be disposed in the detection path (see below) between the elements 30 and 46, particularly in connection with focusing on scattering objects.

Via two beam dividers 30, 32 in the form of dichroic or semi-permeable mirrors, light is transmitted from the light source 12 into the lens 26 of the optical imaging system 4 housed in a microscope housing 34 and the light focuses on the sample 6. For this purpose, the lens 26 comprises a optical element 36, e.g. a lens which can be moved controllably by means of an actuator 38 along the optical axis 20. of the lens 26 The control of the position of the optical element 36 and thus of the focus of the sample 6 is carried out by the control device 10. The actuator itself can comprise an independent distance meter.

Light reflected from sample 6 runs through the lens 26 in the opposite direction, as indicated by a dotted arrow, and is passed through the beam splitter 32 on one side into an optic 40 and into the image detector 8 and on the other side via the beam splitter 30 and a further optic 42 to a detector 44 comprising several detector elements (detection path). The detector elements may be single sensors, e.g. photodiodes, or a grid of sensors. In front of the detector 44 is placed an aperture in the optical system 22 with an aperture 46 which is shaped similar to the aperture of the aperture 18 and is located in the image plane of the optical system 22, and in which is generated an image of the sample 6 and hence of the light source pattern reproduced in sample 6. Aperture 46 may comprise one or more apertures and is hereinafter referred to as aperture 46. Detector 44 provides signals to control device 10 which analyzes these and uses them as control or control input for a control of the actuator 38. The control device can further process the independent distance signal from the actuator 38 and, if desired, use it for the control.

FIG. 2 shows a schematically shown beam path (illumination path) in the autofocus device 2 in two light paths 48, 50 on sample 6. In this embodiment, the light pattern of the light source 12 is reduced to a bright spot that radiates through two openings on the means 28 for division into the light paths 48, 50. At an aperture 18, for example, which produces two bright spots, as shown in FIG. 1, the light from each light point is divided into two light paths 48, 50, as indicated by the means 28 of FIG. First

The light from both light paths 48, 50 is focused in a spot-shaped measuring light focus 52 in sample 6, which may take the form of the light source and e.g. is dot-shaped, is oblong to a slit-shaped light source or has any other shape. Since both the measuring light from the light source 12 and the light for the test examination are passed through the lens 26, the measuring light focus 52 can be in the focus of the camera respectively. the optical imaging system 4 which may be a focus plane. However, it is also possible that the focus light focus 52 is removed from a focus 56 of the camera at a previously known distance 54.

The typical sample 6 comprises a sample holder 58 on which biological sample material 60 is provided, which is covered with a thin transparent cover glass 62. This sample 6 reflects incident light at three interfaces 64, 66, 68, namely the highly reflective air glass. interface 64, at glass sample material interface 66 reflecting substantially less strongly, and at sample glass interface 68, which is not considered in the following, where, for very thin sample materials, the signals form a combination of interfaces 66 and 68 In this connection, the glass sample material interface 66 forms a focus target plane 70, which is described in this first embodiment, and to which the focus light focus 52 is to be brought in by the autofocus method.

The autofocusing method performed for this purpose is described on the basis of Figures 3 - 8. Figures 3-6 show the optical system 22 and lens 26 greatly simplified over sample 6, which is only indicated by interfaces 64, 66. Detector 44 is shown by two detector elements 72, 74 located on both sides of the optical axis 20. In an arrangement similar to FIG. 1 with two point light sources, there would be four detector elements. The aperture aperture 46 in front of the detector 44 is suitably of the same shape as the light source, that is, dot respectively. circular in this embodiment. It is positioned in such a way that it will be asymmetrically displaced with respect to the optical axis 20, the axis 20 being outside the aperture 46, that is, not passing through it.

The two light paths 48, 50, in their portion affecting the sample 6, are shown with thin dots and directed to the target light focus 52 located in the sample holder 58, that is, below the focus target plane 70 which is identical to the interface 66. The different light paths from the different interfaces 64, 66 to the aperture opening 46, respectively. to the detector elements 72, 74 are shown differently. The light path from the main reflex reflected from the strongly reflecting interface 64 is shown with fully drawn lines and the light path from the light reflected from the less reflecting interface 66 by dotted lines. It will be seen that, firstly, no light or only a negligible amount of light is reflected in the measuring light focus 52, and secondly, the light reflected from the interfaces 64, 66 does not hit the aperture 46 so that light does not reach from it to detector elements 72, 74. In FIG. 4, the sample 6 is moved downward in comparison with the image of FIG. 3, as indicated by arrows, such that the target light focus 52 is moved upward relative to the sample 6. The movement of the sample 6 is equivalent to the movement of the lens 26 by the actuator 38. At the position of the sample 6 relative to the lens 26 shown in FIG. 4, the measuring light focus 52 is just below the interface 66. Due to the asymmetry of the aperture 46 relative to the optical axis 20, at this position reflected light emits from the light path 48 through the aperture 46 and hits the detector element 72, whereas light from the light path 50 does not strike. the aperture 46 so that the detector element 74 remains darkened.

By a further movement of the sample 6 downwardly or of the target light focus 52 in the sample 6 upwardly, the target light focus 52 reaches the boundary layer 66 and the focus target plane 70, as shown in FIG. 5. The reflections of the two light paths 48, 50 intersect in the image plane where the aperture and aperture 46 are located. Due to the asymmetric aperture 46 outside the optical axis 20, the two light paths 48, 50 are extensively darkened but not completely due to the flat aperture of the light paths 48, 50. The two detector elements 72, 74 each receive some and equal light and sends a similar signal to the controller 10.

FIG. 6 shows the light paths 48, 50 in yet another downward movement of the sample 6 or of the measuring light focus 52 of the sample 6 in the upward direction. The target light focus 52 exits the boundary layer 66 and approaches the boundary layer 64, so that the reflection of the boundary layer 66, which only reaches the detector element 74, becomes increasingly darkened and the reflection of the boundary layer 64 increasingly affects the detector element 72 through the aperture 46.

The aperture 46 is located in the image plane of the lens 26. Light reflected from the measuring light focus 52 passes through the aperture 46 and, appropriately, equally powerfully from both light paths 48, 50. The aperture 46 is positioned in such a way that light reflected above or below the measuring light focus 52 runs. out of the light paths 48, 50 through the aperture aperture 46 of varying strength. Thus, an equally powerful illumination of the detector elements 72, 74 means that one of the boundary layers 64, 66 lies in the target light focus. In this connection, the aperture is advantageously only so large that light from a boundary layer 64, 66 having a distance of more than 100 μηη to the target light focus 52 cannot escape from any of the light paths 48, 50 through the aperture 46.

By means of the aperture opening 46, a selection of the light from different light paths by optical length can be made. Also, a selection of the light from different light paths can be made according to their different direction towards the detector elements 72, 74. In FIG. 7, the amplitudes A from signal 76 in detector element 72 and signal 78 in detector element 74 are described by a movement of the measuring light focus 52 in the sample 6 as in Figures 3-6 over the time t. In addition, the movement of position 80 in the measuring light focus 52 is in the z direction, parallel to the optical axis 20 of the lens 26, shown over the time period ten correlating with the signals 76, 78. Four times III, IV, V, VI corresponding to the positions 80 in the target light focus 52 of Figures 3, 4, 5 are marked. 6th

For automatic focusing of the sample 6, the light generator 14 is first connected to the autofocus light source 12 and the lens 26 respectively. its optical element 36, which can be moved by means of the actuator 38, is moved to its initial position - in the figures in the direction of the sample 6 all the way down, so that the measuring light focus 52 is within the sample 6 and, where appropriate, within the sample holder 58.

Now the actuator 38 is moved so that the target light focus 52 is completely moved through the sample material 60 and through the focus target plane 70. At the same time, the signals 76, 78 are received from the detector elements 72, 74 and suitably also a position signal from the actuator 38. First, the signal 76 from the detector element 72 rises to fall quickly again. Thereafter, the signal 78 rises from the detector element 74 and also falls again, both in accordance with the light incident through the aperture 46 as described in Figures 4-6. In particular, the position of the intersection of the flanks is taken from the signals 76, 78 - in the following target position, in which the target light focus 52 is in the focus target plane 70. This target position is recorded by the control device 10, which is connected to the actuator 38, which is constantly or on demand from the control device 10. sends its position respectively. the position of the optical element 36 to the controller 10.

The new sharp increase first of signal 76 and then of signal 78 above a limit value g is considered as a sign and indication that the target light focus 52 approaches the strongly reflective interface 64 and thus is above the focus target plane 70. The upward movement of the target light focus 52 is stopped. .

Now the actuator 38 can be set in a single method step corresponding to the recorded target position and the sample 6 is focused very quickly. The target light focus 52 is set to the focus target plane 70 and thus also the focus of the microscope 4 when the target light focus 52 is in this focus. Otherwise, the focus is set to a desired plane removed from the focus target plane 70 by a known distance.

Accurate focusing is achieved when the movement of the measuring light focus 52 is reversed and the measuring light focus 52 this time is introduced more slowly into the sample material 6, as shown in FIG. 7. Again, a maximum of the signal 76 is formed, and a setting of the signals 76, 78 for signal compliance leads the focus light 52 into the focus target plane 70.

An alternative method can be started with the target light focus 52 being above the sample 6 and from there being run into the sample 6. The main reflex that first arises from the glass-air boundary layer 64 is clearly recorded. Since the thickness of the cover glass 62 is known, e.g. 170 μιτι, the focus light 52 can be moved downward rapidly with this or a somewhat smaller distance. Then the speed of movement can be reduced and the target light focus 52 is moved further downward until the signals 76, 78 are equally strong.

An adjustment to the target position based on the signals 76, 78 is explained below in the light of FIG. 8. A difference signal 82 is formed from the difference between signals 76, 78, e.g. by subtracting the signals 76, 78 used as the control size with zero throughput 84 as the control target value. In the zero pass 84, the target light focus 52 is in target position 86. Advantageously, the detector 44 is calibrated thereto in such a way that the signals 75, 78 are equal when the target light focus 52 is in the focus target plane 70. If the target light focus 52 is to be a bit outside of the reflective boundary layer 66, an offset can be given to a signal 76, 78, or a signal 76, 78 can be amplified to a greater or lesser extent. Hereby the zero passage 84 is correspondingly displaced in the z-direction. When the ratio of offset or gain to offset is known, the focus target plane 70 can be adjusted correspondingly around the interface 66 without changing the autofocus method described in Figures 7 and 8. The corresponding setting of the detector 44 can be performed as a calibration before an autofocus method or during the autofocus method according to the corresponding order of the controller 10.

After setting or adjusting the focus position, the light generator 14 can be switched off and the focus position can be controlled or retained by the position signal of the actuator 38. This has the advantage that the autofocus light pattern is not reproduced with the camera during lighting. Optionally, the light generator 14 can remain connected continuously and the control is performed according to the difference signal 82.

Now images of the sample 6 or 6 can be recorded. of sample 60, if any. at multiple z positions. These can be actuated by a corresponding control of the actuator 38. It is also possible to obtain these by a signal displacement of one or both signals 76, 78.

For recording multiple images of a large sample 6, it is moved in the x-y direction 88, that is perpendicular to the s-axis, respectively. the optical axis 20, as indicated in FIG. 9. The filing can be preserved in this regard. However, if sample 6 is skewed, the measurement light focus 52 is displaced by a distance 90 in the z direction within sample 6. In order to detect this, signals 76, 78 are checked at the new x-y position for plausibility. If the signals 76, 78 do not match the expectation, that is, outside the limit values, a rough search for the focus target plane 70, as described in FIG. 7. If the signals 76, 78 are acceptable, the control can be started immediately, e.g. for zero examination 84.

FIG. 10 shows a projection of a light source pattern on a resting, skewed sample 6.

It is not possible, on the basis of a single autofocus bright spot, to see if the sample 6 is skewed relative to the optical axis 20. However, if the target light focus 52 includes multiple focus points 92, e.g. as the light pattern is reproduced at multiple focal points 92 in the sample, reflections from each focal point 92 can be analyzed separately via at least two light paths, as described above, in each case. In this way, it can be recorded that the respective focus target plans for the individual focal points 92 are not identical. An error signal may be output so that sample 6 is again inserted straight into its holder.

FIG. 11 and 12 show alternative detection schemes employing two optical paths that are not spatially separated in the optical system 22. In FIG. 11, a beam in the detection path is first separated by the optical system 22 and before the detectors 72, 74 by a semi-permeable mirror 94. By means of two aperture apertures 46 located asymmetrically in relation to the mirror 94 in front of the detectors 72, 74, the distance signal is detected. from the slightly different lanes. The asymmetry is shown at the different distances 96, 98 at the aperture openings 46 perpendicular to the mirror 94. In FIG. 12, the light generator 14 emits radiation at two different frequencies (λι, \ z) divided before the detectors 72, 74 by a dichroic mirror 200. Again, the distance signal is generated by the aperture 46. In this case, the apertures 46 may be located symmetrically in relative to the dichroic mirror 100, when the refractive index of optical system 22 sufficiently divides the light paths of the different frequencies spatially, as shown in FIG. 12 at the distances of the two light paths to the mirror 100.

Also in the exemplary embodiments of FIG. 11 and FIG. 12, by means of an optical means, in these cases the semi-permeable mirror 94 or the dichroic mirror 100, a selection of the light from different light paths by optical length can be made. A selection of the light from different light paths can also be made according to different directions to the detector elements 72, 74.

FIG. 13 is a schematic representation of an optical imaging system 102, e.g. is designed as a microscope and directed to a sample 106 lying on a sample table 104. The optical imaging system 102 comprises a light source 108 whose light rays in a beam indicated by a fully drawn arrow 110 are directed toward the sample 106 by means of an optical system 112 and a dichroic mirror 114. The optical system 112 includes a lens 116 which can be moved by means of an actuator 18 relative to a microscope housing 120 along the optical axis 122 of the beam path for focusing the sample 106.

Radiation reflected or scattered from sample 106 is conducted in a beam path indicated by a dashed arrow through the dichroic mirror 114 and optical elements 124 only indicated in optical system 112 into a camera 126 which is provided with a detector 128 with a light filter 130. The detector 128 comprises a large number of detector elements 132 which are located in a two-dimensional matrix and which are designed as CCD elements and are attached to a chip. The light filter 130 is a spectral filter with multiple filter regions 134 that reflect differently spectrally, and which is also located on the chip and in the beam passage immediately before the detector elements 132.

The sample table 104 and with this sample 106 can be moved by means of an actuator 136 perpendicular to the optical axis 122 of the lens 16, as indicated by arrows 138, so that multiple recordings of sample 106 can be made at different positions of sample 106 relative to the microscope 102. The actuator 136 can be actuated by means of a control device 140 for the microscope 12 in such a way that a movement path of the sample 16 can be adjusted from recording to recording to a predetermined or calculated value of the control device. The control device 140 can simultaneously be the control device 140 for the camera 126 or an additional control device for the microscope 102 outside the camera 126.

Alternatively to or in addition to the actuator 136, an actuator 142 can be activated for the light filter 130 and / or an actuator 144 for the detector 128 by means of the control device 140, so that the filter regions 134 and / or the detector elements 132 can be moved controllably with respect to the optical system 112 perpendicular on the optical axis 122 of the beam path affecting the camera 126. In this way, an image of an object in the sample 16 can travel in one or more ways via the light filter 130 and / or the detector 128.

An alternative embodiment of a detector 146 having multiple detector regions 148, 150, 12 is shown in FIG. 14. The following description is essentially limited to the differences over the embodiment of FIG. 13, with reference to unchanged features and functions. Components that are essentially the same are numbered with the same reference numerals and the aforementioned characteristics are not taken over without being re-described.

Via two dichroic radiation dividers 154, 156, radiation reflected from sample 6 is divided into three spectral regions and directed toward the detector regions 148, 150, 152. The detector regions 148, 150, 152 are each only sensitive in one of the spectral regions or more sensitive here than in the other spectral ranges. Prior to each detector region 148, 150, 152, in each case, a filter region 158, 160, 162 is located, the filter regions 158, 160, 162 being permeable only in one of the spectral regions or more permeable here than in the other spectral regions. Their permeability is spectrally matched to the detector regions 148, 150, 152 assigned to them in each case. One or all of the filter regions 158, 160, 162 may again be subdivided into sub-regions, each of which filters differently spectrally, as shown in FIG. 14. Due to the division into detector regions 148, 150, 152 with different spectral sensitivity and with filter regions 158, 160, 162 each filtering differently spectrally, a particularly high light output can be obtained over a wide spectral range. Figures 15 - 18 show in each case a reproduction of a sample 106, provided with 3x10 test tubes, which can also constitute test surfaces, 164, also considerably larger numbers can be imagined, which in each case contain the same or a different test substance. The test tubes 164 are placed in a square matrix and secured to the sample table 104. In the test substance there are objects 166 to be examined. Sample 106 shows in its entirety and in its objects a pictorial pattern.

The detector 128 and its 11 x 15 square detector elements 132 are, for the sake of clarity, shown in dotted lines, whereas the light filter 130 with its 5 strip-shaped filter regions 134 is shown in solid lines. The stripes in the filter regions 134 are positioned perpendicular to the direction of movement of the sample table shown by an arrow 138. In order to better distinguish the lines from one another, the representation of the sample is shown in broken lines.

FIG. 15 shows the detector 128 and the reproduction of the sample 106 in a mutual position in which a recording of the sample 106 is made, but not the first one, as explained below. The recording constitutes an image section with five image areas, in which twelve sample 164 and contents are shown completely and three sample 164 are only partially shown. Three test tubes 164 are reproduced in each case through a filter region 134 and thus in its spectral range. One in five image areas of the image section is reproduced in each case through a filter area 134. Each filter region 134 and each image area in this connection covers exactly three detector elements 132 perpendicular to the direction of movement of the sample 106, more generally: exactly an equal number of detector elements 132.

For a subsequent recording, the reproduction of sample 106 is further moved by a distance corresponding to the width of filter regions 134, the width being seen in the direction of movement of sample 106. An additional image section of sample 106 is now recorded, this image section covering another sample section and other objects 166. The location of filter regions 134 in the image sections remains the same, but not relative to the sample sections and objects 166. With the second recording, they are reproduced on new reproduced sample glass 164 in a different spectrum, that is, in a different color.

FIG. 17 shows the sample 106, which is again offset by the width of the filter regions 134, so that the test tubes, now reproduced three times, are reproduced in three different spectra. In this way, all regions of sample 106 and all sample tubes 164 are reproduced at least as often as filter regions 134, in the illustrated embodiment at least five times, so that each sample region is recorded in five spectra. Based on these five images, a five-color image can be compiled for each sample area. To reproduce each sample region five times, at the first recording, sample 106 is taken through only one filter region 134, at the second recording through two filter regions 134, and so on. FIG. 15 thus shows the fifth recording of sample 106.

The sample 106 is reproduced in its entirety, the sample 106 being reproduced in section on the detector 128, and several images partially covering each other, of the sample 106 and the objects 166. At least as many images as there are various images are performed. filter sections 134. From as many images covering each other as there are different filter sections 134, in each case a multicolored image of the sample 106 and 106 respectively is produced. of an object 166, e.g. of the control device 140.

The recordings are analyzed in the interconnection of an analyzing device, which may be the control device 140 which is connected signal-to-detector 128. For example, it is recorded, for example, that an object 168 is of particular importance and should be displayed in a particularly high resolution. When this task is defined, the sample 106 is moved from recording to recording by only less than one pixel length, that is, a length of a detector element 132, as can be seen in FIG. 18 in comparison with FIG. 17. In this connection, the object is moved subpixelically from recording to recording via a boundary between two filter regions 134. From the recordings, a resolution located in the subixel area can be obtained so that the object 168 can be displayed. with particularly high resolution.

Alternatively, to move the sample 106 relative to the microscope 102, the light filter 130 and / or the detector 128 may be moved relative to the sample 106 and, for example, the microscope housing 120. In a further embodiment, the charge of the individual detector elements within a filter region with the sample image may be displaced detector elementally and first selected after one or more offsets. Or the charges assigned to a sample position during the displacement of the sample image within a filter range may be assigned a pixel spectral value. In this way, the charge generated by the light can accumulate from the sample over a longer period of time.

FIG. 19 shows a double diagram in which the filter surface of a light filter 170 is shown in the x and y directions. the z-direction is the direction of the optical axis 22 upon entering the camera 126. In addition, the absorption A at the light filter 170 is shown. The higher the transmission of the light filter 170, the lower the absorption A. In the shaded region, the absorption is ideally almost 100% and the light filter 170 is not light permeable.

The light filter 170 is an edge filter with an edge 172 at a fixed wavelength λ. The wavelength λ depends on the position of the edge 172 in the x-direction 170 of the filter 170. Further to the right of the filter, the wavelength λ of the edge 172 is larger than farther to the left. In the example shown, the change of the wavelength of the edge is constant per second. stretch light filter in x direction. Other relationships with linear or non-linear change may also be envisaged. At the light filter 170, a very large number or an infinitely large number of filter regions filtering differently spectrally, very closely or infinitely close together. When the light filter 170 is used instead of the light filter 130 in Figures 15 - 18, each sample area can be reproduced as often as desired in different spectra, so that a spectral resolution of the overall image of sample 106 is dependent on the range of motion of sample 106 from recording to recording. In this way, the spectral resolution of the overall image can be freely chosen.

If two edge filters 174, 176 are placed with an opposite edge sequence one after the other, as shown in FIG. 20, a transmission window 178 can be set both in its spatial extent Δχ and in its spectral extent Δλ by a movement of the edge filters 174, 176 relative to each other. Spectral ranges can be excluded and a spectral resolution can be set.

An adaptation of the light filter 130 to the detector 128 is shown in FIG. 21. In FIG. 21, a graph of the sensitivity E at detector 128 over the wavelength λ of the recorded light is plotted. Sensitivity E is dependent on the wavelength of light λ and one wavelength λι less than a wavelength K2. In order to obtain as uniform illumination of the recordings of the sample 106 as possible over the entire relevant spectral range, at the wavelength λι transmissive filter region 134 of the light filter 130, it is transmissive in a larger wavelength range Δλι than the wavelength hz transmissive filter region 134 of the light filter 130, which is transmissive in a smaller wavelength range ΔΚζ.

A further possibility of obtaining as uniform an illumination as possible of the recordings of the sample 106 over the entire relevant spectral range consists in performing an electronic adaptation of the detector elements 132 to the filter region 134 which lies in front of them. At a smaller transmissive filter region 134, a detector element 132 assigned to this filter region 134 can be activated in a different way than a detector element 132 assigned to a more transmissive filter region 134. The different activation can be achieved by a different setting of the gain factor and / or detector elements 312's integration time.

Also a pixel binding, ie merging two or more pixels respectively. detector elements 132 can be thought of, just like a subsampling, that is, a selection of only every nth detector element 132, where n = 1, 2, 3, ... The corresponding control can be performed by the control device 140. In a particularly advantageous embodiment, the electronic adjustment of the detector elements 132 takes into account a displacement of the filter regions 134 in front of the detector elements 132. For this, the position of the light filter 130 relative to the detector 128 must be known, e.g. using position signals from one of the actuators 142, 144. ....... 68 Interface

Reference designations 70 Focus target plane 2 Autofocus device 72 Detector element 4 Microscope 74 Detector element 6 Sample 76 Signal 8 Image detector 78 Signal 10 Control device 80 Position 12 Light source 82 Difference signal 14 Light generator 84 Zero passage 16 Optical 86 Target position 18 Aperture 88 Direction 22 Optical axis 90 Stretch Optical element 94 Mirror 26 Lens 96 Distance 28 Medium 98 Distance 30 Beam parts 100 Mirror 32 Beam parts 102 Microscope 34 Microscope housing 104 Sample table 36 Optical element 106 Sample 38 Actuator 108 Light source 40 Optics 110 Arrow 42 Optics 112 Optical system 44 Detector 114 Mirror 46 Aperture 116 Lens 48 Light path 118 Actuator 50 Light path 120 Microscope housing 52 Target light focus 122 Optical axis 54 Distance 124 Optical elements 56 Focus 126 Camera 58 Sample holder 128 Detector 60 Sample material 130 Light filter 62 Cover glass 132 Detector element 64 Interface 134 Filter range 66 Interface 136 Actuator 138 Arrow 168 Object 140 Control unit g 170 Light Filter 142 Actuator 172 Edge 144 Actuator 174 Edge Filter 146 Detector 176 Edge Filter 148 Detector Area 178 Transmission Window 150 Detector Area A Absorption 152 Detector Area E Sensitivity 154 Mirror λ Wavelength 156 Mirror Δλ Wavelength Range 158 Filter Range A Amplitude 160 Filter Range g Limit 162 Filter Range t Time 16 The direction of the optical axis 166 Object

Claims (12)

An autofocusing method in which light from a light source (12) is divided into two light paths (48, 50) through two openings on a means (28), focused in a measuring light focus (52) in a sample (6) and thrown back therefrom, and the reflected light is passed through an optical system (22) in two corresponding light paths (48, 50) to at least two detector elements (72, 74), where the target light focus (52) is moved in layers that reflect light of different strength, of the sample (6); and the reflected light is passed through at least one aperture aperture (46), which in its shape corresponds to the shape of the light source (12), is located in an image plane of the optical system (22) and is located outside an optical axis (20). ) in the optical system (22); and the detector elements (72, 74) are arranged in such a way that the course of a radiation property detected by one of the detector elements (72, 74) is mutually different and a focus position is set depending on the paths. Method according to claim 1, characterized in that light reflected from the target light focus (52) from the two light paths (48, 50) strikes the detector elements (72, 74) with the same strength. Method according to one of the preceding claims, characterized in that light reflected above or below the target light focus (52) strikes the detector elements (72, 74) of different strength. Method according to one of the preceding claims, characterized in that, by means of an optical means, a selection of the light reflected from different boundary layers (64, 66) is performed according to different optical path length. Method according to one of the preceding claims, characterized in that, by means of an optical means, a selection of the light reflected from different boundary layers (64, 66) is carried out in different directions towards the detector elements (72, 74). Method according to one of the preceding claims, characterized in that the light paths (48, 50) are separated from each other by a shaded area. Method according to one of the preceding claims, characterized in that the light in the light paths (48, 50) has different spectral properties and the light paths (48, 50) are divided before the detector elements (72, 74) according to the spectral properties. Method according to one of the preceding claims, characterized in that the processes are recorded continuously. Method according to one of the preceding claims, characterized in that a focus in the optical system (22) is set in such a way that signals (76, 78) from the detector elements (72, 74) are in a fixed relationship with each other. and especially has the same strength. Method according to one of the preceding claims, characterized in that the measuring light focus (52) is set to a boundary layer (66) which reflects light and that the sample (6) is then moved perpendicular to the optical axis (20) of the optical system. (22) and the signals (76, 78) from the detector elements (72, 74) are subsequently checked for plausibility with respect to a continued coarse setting to the reflective boundary layer (66). Method according to one of the preceding claims, characterized in that the light source (12) contains a light pattern reproduced in the sample (6), in each case reflected light from several pattern points in the light pattern being recorded separately according to light paths (48, 50 ). An autofocus device (2) comprising: a light source (12); a means (28) having two openings for dividing the light from the light source into two light paths (48, 50); a lens (26) for focusing the divided light in a measuring light focus (52) in a sample (6) and for directing the reflected light therefrom in two corresponding light paths to at least two detector elements (72, 74); and an optical system (22) for imaging the light source pattern produced in the sample on an image plane of the optical system (22); at least one aperture aperture (46) corresponding in shape to the shape of the light source (12) located in said image plane and outside an optical axis (20) of the optical system (22); an actuator (38) and a control device (10) for moving an element (36) of the lens (26) relative to the sample (6) through the actuator (38) in such a way that the target light focus (52) is moved in layers, reflecting light of different strength from the sample (6); in such a way that the reflected light is passed through the at least one aperture aperture (46), wherein the detector elements (72, 74) are positioned in such a way that a radiation property detected by one of the detector elements (72, 74), in this connection are different and the control device (10) is adapted to analyze the processes at several positions of the target light focus (52).